Defects of Tfn recycling in Sac2 null cells. (A) Western blot analysis of biotin surface-labeled TfnR. (B) Quantification was performed as in Fig. 4 B. Values are normalized to WT cells (surface/total). Data are from three replicate experiments (mean ± SEM). (C) Flow cytometry analysis of Tfn recycling in WT and Sac2 null N2A cells. Data are from three replicate experiments (n = 5,000 in each time point; mean ± SEM). (D) Representative images of Tfn recycling at the indicated times points. (bottom) Cells transfected with Flag-Sac2 (green). Bars, 10 µm. *, P < 0.05; **, P < 0.01.